Project Summary/Abstract: Vitamin B12 (cobalamin) deficiency is a significant public health problem among the elderly. Deficiency can lead to a wide spectrum of hematological (pernicious anemia) and neurological disorders (dementia) which are usually reversible by early diagnosis and prompt treatment. The primary causes of deficiency is malabsorption due to a lack of intrinsic factor and gastric atrophy. Accurate diagnostic tests for vitamin B12 malabsorption are needed because the symptoms attributable to mild vitamin B12 deficiency, neurological dysfunction and dementia, are non-specific and difficult to diagnose. The currently used procedure for assessing B12 malabsorption is the Schilling Urinary Excretion Test, first introduced in 1953. While this radioisotopic test has made great advances in understanding vitamin B12 related disease, it is imprecise and cumbersome (requires 24 hour urine collections). As a result, the underlying causes of B12 deficiency are frequently not addressed, despite the chronic and degenerative aspects of the problem. The market for a simple replacement has been estimated at up to 1,000,000 tests per year in the continental USA, although only an estimated 40,000 Schilling tests per year are currently administered. This proposal describes a new absorption test method based upon a single finger-prick measurement that can be applied to pediatric populations as well as adults. The working title for the new assay is VSI-B12. The test is made possible by 2 key technological innovations: an inexpensive biosynthetic system for production of isotopic (14C) vitamin B12 (dubbed tracer B12), and an ultra sensitive detection of the tracer B12 by Accelerator Mass Spectrometry, a tool that achieves zeptomole (10-21) sensitivity towards C isotopes, 14 the lowest levels of any known analytical instrument. Using this approach, the absorption of an oral dose of Tracer B12 can be quantified in a single drop of blood using harmlessly small radioactive doses (25 nanoCuries). For Phase 1, we propose a plan for optimization of the bacterial vitamin B12 labeling system so as to more efficiently produce tracer B12 for further clinical evaluation (Phase 2). Specific Aims of this proposal are: 1. Perform a set of experiments to optimize biosynthesis of Tracer B12 from our engineered bacterial system. 2. Biosynthesize and purify 100 test doses to be administered orally in Phase 2. 3. Identify clinical PI's who have experience with the Schilling test and willingness to serve as collaborative partners in the development of VSI-B12 The work has significance as it should lead to a convenient diagnostic test for the clinical B12 malabsorption that can be employed in the clinic or in research settings. The format described, once successfully introduced to the research community, should stimulate similar development in other diagnostic areas, and could usher in a new era for tracer diagnostics where minute quantities of an appropriate 14 C probe are quantified by Accelerator Mass Spectrometry from microliter-sized specimens. Project Narrative: This proposal describes a new test method made possible by 2 key technological innovations: an inexpensive biosynthetic system for production of isotopic (14C) vitamin B12 (Tracer B12), and an ultra sensitive detection of the Tracer B12 by Accelerator Mass Spectrometry. The work is significant as it would lead to a needed convenient diagnostic test for the clinical B12 malabsorption that can be employed in the clinic or in research settings. [unreadable] [unreadable] [unreadable]